The growth of Escherichia coli cultures under the influence of pheomelanin nanoparticles and a chelant agent in the presence of light

Growing concern of antibiotic resistance has increased research efforts to find nonspecific treatments to inhibit pathogenic microorganisms. In this regard, photodynamic inactivation is a promising method. It is based on the excitation of a photosensitizer molecule (PS) with UV-Vis radiation to produce reactive oxygen species. The high reactivity of such species nearby the PS leads to oxidation of bacterial cell walls, lipid membranes (lipid peroxidation), enzymes, and nucleic acids, eventually producing cell death. In the last decade, many studies have been carried out with different photosensitizers to suppress the growth of bacteria, fungi, viruses, and malignant tumors. Here, our main motivation is to employ pheomelanin nanoparticles as sensitizers for inhibiting the growth of the Gram-negative bacteria E. coli, exposed to blue and UVA radiation. In order to perform our experiments, we synthesized pheomelanin nanoparticles from L-DOPA and L-cysteine through an oxidation process. We carried out experiments at different particle concentrations and different energy fluences. We found that cultures exposed to UVA at 166 μg/mL and 270 J/cm2, in conjunction with ethylenediaminetetraacetic acid (EDTA) as an enhancer, decreased in the viable count 5 log10. Different reactive oxygen species (singlet oxygen, hydroxyl radicals, and peroxynitrates) were detected using different procedures. Our results suggest that the method reported here is effective against E. coli, which could encourage further investigations in other type of bacteria.


Supporting information
Based on the oxidation method proposed by Pyo et al. [1] we present below the details of the pheomelanin nanoparticles synthesis used in our research work. We also show the results of the characterization with different methods.

Synthesis
For the synthesis of pheomelanin particles, 70 mg (0.35 mmol) of 3,4-dihydroxyL-phenylalanine (L-DOPA, D9628, Sigma-Aldrich) was dissolved in deionized water (milli-Q, 18.2 MΩcm) at 50 o C. The solution was then brought to room temperature. Next, 85.6 mg (0.70 mmol) of L-Cysteine (C7352, Sigma-Aldrich) was added to the solution followed by 360 µL of KMnO4 (1 N, 223468, Sigma-Aldrich). The referred process was carried out under continuous stirring at 1500 r.p.m during 24 h and dark conditions. The obtained particles were precipitated by centrifugation for 10 min at 20,000 r.p.m. They were resuspended in 50 mL of deionized water and mixed with 1 mL of HCl (1 M) to exchange the Mn 2+ ions in the solution. In order to raise the pH of the sample, the suspension was subjected to six washing cycles at the formerly described conditions. A last centrifugation (10 min at 4500 r.p.m) was carried out to discard big aggregates. The final value of pH was 6. The resulting nanoparticles were resuspended in PBS in order to obtain a stable medium for the cell culture. The solution, now with pH 7.4 was stored in an amber glass jar at 4 o C.

UV-Vis spectroscopy
The synthesis process was monitored by assessing the change of the maximum absorbance peak from the precursor L-DOPA (at 280 nm) using a spectrophotometer (Multiskan, GO Thermo Scientific). Once the pheomelanin particles were obtained, 1 mL of the solution was poured into a polystyrene cell and the absorbance spectrum from 200 to 1000 nm was obtained.

Lyophilization
Pheomelanin solution was lyophilized using a Labconco FreeZone 2.5 system. The solution was first centrifuged at 20,000 r.p.m to remove water excess. The samples were liophylized at a pressure and temperature < 0.133 mBa and -40 o C and stored at -20 o C between 1 and 3 days before characterization.

Dynamic light scattering
Particle sizes and zeta potentials were determined by dynamic light scattering (DLS) using a Malvern Zetasizer NanoZS equipment. See main text for details.

Fourier transform infrared spectroscopy
The functional groups present in the synthesized samples allow us to assess the purity of the pheomelanin particles. The samples were evaluated using a FTIR spectrometer (Agilent Cary 630) in the transmittance mode. The powder was placed on the ATR accessory (DATR Agilent) made of diamond crystal (R.I=2.4). The spectrum was obtained in the range 4000-650 cm -1 , at a path length of 2.6 µm and room temperature. A total of 32 scans were performed with step of 4 cm -1 . Every experiment was carried out ten times using different samples. All spectra were background subtracted and smoothed with 7-point Savitsky-Golay function to reduce the noise. The data were analyzed using the Origin 2016 software.

X-ray diffraction analysis
X ray diffraction (XRD) patterns of the samples were obtained on a XRD system (Bruker D-8 Advance) using CuKα radiation. The instrument was operated at 30 mA and 40 kV. The scans were performed between 3-60 o 2θ, using a step size of 0.02 and dwell time of 0.5 s. The experiment was performed seven times. Data acquisition and evaluation were performed by the Diffract.suite software package.